The extensive use of insecticides, during the past decades has led
to a number of negative effects on terrestrial and aquatic organisms.
Chlorfenapyr is a member of a new class of chemicals, namely, pyrroles
(chemical name: 4-bromo-2-(4-chlorophenyl)-1-(ethoxymethyl)-5-(trifluoromethyl)- 1H-pyrrole-3carbonitrile; trade name: Pylon
miticide-insecticide). Chlorfenapyr uncouples oxidative phosphorylation
in the mitochondria, resulting in disrupted ATP production, cellular
death, and ultimately, death of the organism [8].

Chlorfenapyr, which is a member of the pyrrole class of chemicals,
is a proinsecticide compound; that is, its biological activity depends
on its activation by another chemical. Oxidative removal of the
N-ethoxymethyl group of Chlorfenapyr by mixed function oxidases forms
uncouples oxidative phosphorylation at the mitochondria, resulting in
disruption of production of ATP, cellular death, and ultimately organism
mortality [3].

Monitoring of aquatic ecosystem pollution represents one of the
major activities involved in measures aimed at environmental protection.
Usage of non-targeted organisms in environmental toxicology is needed to
understand the wide range of toxic effects caused by the pesticides on
different organisms [23]. Fish and other aquatic biota that were
commonly used as bioindicators of persistent organic pollutants [22]
have been replaced in recent years successfully by ciliates [12].
Protozoan cells are often used as bioindicators of chemical pollution,
especially in aqueous environment. The application of unicellular
organisms to study the toxic effects of pesticides from contaminated
wastewater is relatively new throughout the world. Hence, in the present
paper, we have studied the toxic impacts of Chlorfenapyr a novel
broad-spectrum insecticide-miticide, stable and persistent in the
environment on oxidative stress in a pure culture of Paramecium sp.

MATERIALS AND METHODS

The biological material used is the typical representative of
Protozoa: Paramecium sp. We kept a continuous pure colony in the
laboratory at the University of Annaba from infusions of lettuce [1].

The pyrrole 4-Bromo-2-(4-chlorophenyl)-1-(ethoxymethyl)-5-(trifluoromethyl)-1H- pyrrol-3-carbonitrile is the common name of Chlorfenapyr
that is used as an acaricide/insecticide and developed by Dow
AgroSciences. This synthetic molecule was tested at the following
concentrations: 250, 300 and 350 [micro]M. These concentrations were
selected after a series of preliminary finding range toxicity tests.

In accordance with Lavergne [9] and Sauvant, Pepin and Piccini
(1999), the growth kinetics of Paramecium sp. were studied by measuring
the optical density OD at [lambda] = 600 nm in function of time.

The response in percentage was calculated to evaluate the toxicity
of xenobiotics via the inhibition of cell growth of protists. The
percentage of positive values indicates an inhibition of growth, while
the negative values indicate a stimulation of growth [26].

The polarographic method enables measurement of production or
consumption of oxygen with an oxygen electrode, type HANSATECH [11].

The determination of glutathione (GSH) was performed as described
by Weeckbeker and Cory [24] and glutathione S-transferase (GST) by
Habig, Pabst and Jakoby [7]. These biomarkers were chosen for their role
in cell protection and in cellular metabolism of xenobiotics.
Measurements were made during the exponential phase of growth (between
48 and 96 h).

All the experiments were repeated three times, and the results were
expressed as mean and standard error (SE) values. Statistical analysis
was performed using a two-way ANOVA and the test of Dunnett for
comparison between the control and treated cells. The [alpha]-level for
significant differences was set at p<0.05 [5].

Results:

Effect of Chlorfenapyr on the growth of Paramecium sp:

The impact of Chlorfenapyr concentrations on the population growth
of Paramecium sp. is shown in (Figure 1). The Chlorfenapyr has an
inhibitory effect on cell growth of Paramecium (p<0.001), and this
effect was already visible at 2nd day of the treatment.

Percentage of response in Chlorfenapyr treated Paramecium sp:

The response percentage measurement results are presented in
(Figure 2). It can be said that the positive evolution of the response
percentages confirm the growth inhibition of the treated paramecia and
this regardless of the cell concentration.

Effect of Chlorfenapyr on GSH level in Paramecium sp:

As shown in figure 3, the GSH level is lower at the concentrations
of 250 and 300 gM than in the nontreated Paramecium. In contrast, with
the highest concentration (350 [micro]M), the GSH level was increased
significantly (p <0.001). We hypothesize that this increase results
from the started detoxification/metabolism process.

Effect of Chlorfenapyr on GST activity in Paramecium sp:

As demonstrated in figure 4, that GST activity increased
significantly in cells treated with different concentrations of
Chlorfenapyr compared to controls (p <0.001).

Effect of Chlorfenapyr on the respiratory metabolism of Paramecium
sp:

The [O.sub.2] consumption of paramecia was significantly affect
(p<0.001) by the action of Chlorfenapyr concentrations (Figure 5). It
should be noted that after 5 min of exposure, cell cultures treated with
strongest concentration (350 [micro]M) present a significantly
deceleration of their respiratory activity.

Discussion and conclusions:

Paramecium that has long been a model organism for cellular aging
and clonal lifespan [18,17,16,15,20,21].

In the present study, we showed that Chlorfenapyr concentrations
molecule caused a dose-dependent growth inhibition of Paramecium sp.
population. In addition, for the three concentrations tested, it can be
said that the evolution of the response percentages confirm the growth
inhibition of the treated Paramecium and this regardless of the cell
concentration.

Low level of GSH and high GST activities recorded in the presence
of xenobiotics may be indicative of activation of the detoxification
system. On the other hand, as reported before [14,27,10,6] the direct
capture of oxygen free radicals caused by xenobiotics is carried out by
radical compounds as traps or enzyme systems located nearby the initial
glutathione production system.

Chlorfenapyr is a member of a new class of chemicals--the pyrroles.
The compound is a pro-insecticide, i.e. the biological activity depends
on its activation to another chemical. Oxidative removal of the
N-ethoxymethyl group of Chlorfenapyr by mixed function oxidases forms
uncouples oxidative phosphorylation at the mitochondria, resulting in
disruption of production of ATP, cellular death, and ultimately organism
mortality [3]. This inhibitory effect is reflected in our work by a
metabolic disorder in the cellular respiration generated by an oxidative
stress [4,25].

The results suggest that the antiproliferative effect of
Chlorfenapyr may be mediated by reducing the chitin and cuticle of
cells, also by the effect on mitochondria and lashes, our results allow
us to conclude that the tested Chlorfenapyr inhibits the growth of
Paramecium, and that this effect is mediated by a disruption of its
cellular metabolism.

After considering all the experimental data obtained throughout the
study, it appears that the ciliate protists used in our work is a
material of choice for studies in toxicology and occupies a privileged
position in aquatic ecosystems because it is one of the basic elements
of food chain, hence the need for a deep study of the impact of
pollution on our environment.